Cystic fibrosis (hereinafter referred as CF) is characterized by the presence of highly viscous pulmonary secretions in the lung (Wine in J. Clin. Invest. (1999) 103:309-312). The origin of these secretions, also described as mucus, which covers the epithelial cells of the bronchi and upper airways, has been associated with a mutation in the CFTR (i.e. Cystic Fibrosis Transmembrane conductance Regulator) gene (Collins in Science (1992) 256:774-779). CFTR codes for a protein, which functions as a chloride channel in the apical membrane of epithelial cells of the lung and intestine. In the lung this leads to a decreased chloride flux from the epithelial cells into the respiratory mucus layer. This, combined with an increased sodium and water absorption from the mucus into the cells (Knowles in N. Engl. J. Med. (1981) 305:1489-1495) probably results in an enhancement of the mucus viscoelasticity. Additionally, it is hypothesized that the gene defect in CF may also lead to altered mucins which bind bacteria more tightly (Scharfman et al. in Infect Immun. (1996) 64:5417-5420).
Due to the impaired clearance of inhaled pathogens, chronic bacterial colonization of the lungs are very common in CF patients. These lung infections evoke a migration of leukocytes and neutrophils into the mucus. However, these inflammatory cells, together with the pathogens and epithelial cells also die and release nuclear DNA and actin in the mucus, which further enhances the viscoelasticity and the decreased clearance of the mucus. DNA concentration in the mucus of CF patients typically ranges from 1-15 mg/ml (Shah et al. in Respir. Med. (1995) 89:499-502; Zahm et al. in Eur. Respir. J. (1995) 8:381-386; Vasconcellos et al. in Science (1994) 263:969-971; Sanders et al. in Am. J. Respir. Crit. Care Med. (2001) 164:486-493).). The high viscosity of mucus also causes suffering and even morbidity in diseases like chronic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis.
Some of the compounds in mucus (DNA and actin) are released from leukocytes that infiltrate pulmonary tissue in response to the presence of micro-organisms, such as Pseudomonas, Pneumococcus and Staphyloccus bacteria, or other irritating factors such as pollen and smoke.
In order to facilitate the removal of lung secretions, mucolytic drugs able to decrease the mucus viscoelasticity are often used. The viscosity of mucus of CF patients is significantly reduced by the administration of DNA degrading proteins. The protein most frequently used is DNAse I, an endonuclease that hydrolyses the highly viscous double stranded and single stranded DNA preferentially at a site adjacent to pyrimidine nucleotides, resulting in shorter oligonucleotides with decreased viscosity. In addition, surfactants in the sputum are released when the DNA network is degraded.
DNAse I has an optimal activity at a pH between about 5.5 and 7.5, preferably near pH 7.0 (Shak et al. (1990) Proc. Natl. Acad. Sci USA 87, 9188-9192). The presence of divalent cations, such as magnesium, in the catalytic centre of DNAse I is essential for its activity. In the presence of Mg2+ the cleavage at both strands occurs independently from each other. In the presence of manganese (Mn2+) ions, cleavage occurs at approximately the same site, resulting in blunt or nearly blunt ended fragments.
The human homologue of human DNAse I has been purified, cloned and expressed as a recombinant protein and is approved as a medicine. Recombinant human DNAse I (hereinafter rhDNAse I) is commercially available under the trade name Pulmozyme and is also designated as “dornase alpha”. Although clinical trials have shown that rhDNAse I significantly improves lung function and decreases exacerbations in some CF patients (Shah et al. in Respir. Med. (2000) 89:499-502; Fuchs et al in N. Engl. J. Med. (1994) 331:637), it does not mean that all patients show clinical improvements. Indeed, other studies claim that there is a wide variation in clinical response of CF patients to rhDNAse I (in particular Bollert in Eur. Respir. J. (1999) 13:107-113; Christopher in J. Clin. Pharm. Ther. (1999) 24:415-426; Cobos in Eur. J. Pediatr. (2000) 159:176-181) and that a significant number of patients (the so-called non-responders) show no benefit from rhDNAse I therapy.
The reason for the failure in these non-responders is not clearly understood. Several variants or alternatives on the rhDNAse I treatment, some of them being relevant to the present invention, are described below.
The high viscosity of the mucus impedes the diffusion and penetration of DNAse inwards the mucus. Some surfactants that also reduce the viscosity may provide additional benefits by increasing the diffusion and subsequent performance of administered DNAse. U.S. Pat. No. 5,830,436 provides an example of an alkylaryl polyether alcohol polymer surfactant used in the treatment of pulmonary diseases.
DNAse I is subject to proteolytic degradation by proteases that are produced by leukocytes. The most predominant protease is elastase. U.S. Pat. No. 6,124,257 describes the administration of protease inhibitory proteins in order to improve the integrity and activity of DNAse I.
The catalytic activity of DNAse can be impeded by a shift in pH due to acidic compounds present in the mucus of patients, especially when pH shifts outside of the optimal pH range of 5.5-7.5. U.S. Pat. No. 5,863,563 describes the administration of a pH-raising buffer as a powder or as a nebulised solution. As a consequence of this buffering, DNAse I is able to perform in an optimal pH environment.
The activity of DNAse I decreases with time due to a chemical automodification of an aspartic acid into an isoaspartic acid. A purification method to remove the inactive modified DNAse I and an alternative storage method to prevent this modification are described in U.S. Pat. No. 5,783,433 and U.S. Pat. No. 5,279,823. Herein, the non-deamidated form of DNAse I is separated from the amidated form by ion exchange chromatography.
A major drawback in the DNAse I treatment of pulmonary diseases is its binding to monomeric actin and its subsequent inactivation. Pulmonary diseases are diseases which affect lung function. Such diseases may result from a defect in a gene or genes associated with lung function, asthma, allergies, an immune or an autoimmune disorder a microbial infection or a mechanical injury to the lungs. Actin is the most abundant protein in nucleated animal cells and constitutes approximately 10 to 20% of the protein of many nucleated cells and 30% of the protein of muscle cells. Actin molecules each bind an ATP (adenosine 5′-triphosphate) or ADP (adenosine 5′-diphosphate) molecule and self-assemble into long filaments during which the ATP is hydrolysed into ADP. Injury to animal tissues results in the release of actin into the extracellular space. Although approximately half of non-muscle cell actin is F-actin (the double-helical, rod-like, filament form of actin which is assembled from G-actin monomers). The ionic conditions of extracellular fluids are expected to favour actin polymerisation, so that virtually all the actin released from dying cells would polymerise into filaments if sufficiently concentrated (i.e. greater than a few micrograms per millilitre) as disclosed by Lind et al. in Am. Rev. Respir. Dis. (1988) 138:429-434). In purified solutions, in the absence of filament-shortening proteins, actin filaments can easily attain lengths of several microns. A wide variety of factors that influence the equilibrium between monomeric G-actin and polymeric F-actin are known. A non exhaustive list of these includes toxins (e.g. phalloidin), numerous proteins (lysozymes, kinases, actin related proteins, actin binding proteins) and a variety of ions (potassium, magnesium, cadmium, lithium, nickel, ammonium) as disclosed by Higgs et al. in J. Biol. Chem. (1999) 274:32531-4; Richard et al. in Int. Microbiol. (1999) 2:185-94; Sun et al in Curr. Opin. Cell. Biol. (1995) 7:102-110; Carlier et al. in Adv. Exp. Med. Biol. (1994) 358:71-81; Estes et al. in Cell Motil. (1992) 13:272-284; Shu et al. in Biochem. J. (1992) 283:567-573; Pollard in Curr. Opin. Cell. Biol. (1990) 2:33-40 and Korn in Physiol. Rev. (1982) 62:672-737. The interconversion from G actin to F actin is promoted by Mg2+ and K+ ions (Shu et al. in Biochem. J. (1992) 283:567-573). The differences in K+ and Mg2+ concentration in the two groups (i.e., responders and non responders on DNAse I) are relevant in terms of influencing the polymerisation state of actin. Maximal polymerisation is obtained with concentrations of 2 mM Mg2+ or 200 mM K+ (Shu et al. cited supra).
Due to the large amounts of actin present in cells, the release of actin from dying cells provides sufficient actin to have a significant effect on the microenvironment by increasing the viscosity of extracellular fluids, such as mucus. The lysis of neutrophils is the major source of actin in the mucus of CF patients.
The therapeutic effect of DNAse I on the treatment of diseases such as CF has been attributed to the degradation of the DNA released by the neutrophils resulting in a decreased viscosity. The activity of DNAse I is strongly influenced by the presence of actin. DNAse I binds to G actin after which it is inactivated (Ulmer et al. in Proc. Natl. Acad. Sci. USA (1996) 93:8225-8229). As a consequence of the binding of DNAse I to the actin monomer, DNAse I also acts as an actin depolymerising compound according to Vasconcellos et al. in Science (1994) 263, 969-971. It was suggested that the mucolytic effect of DNAse in pulmonary secretions was rather to actin disaggregating than to DNA hydrolysis. Mutational analysis of DNAse I disproved this hypothesis (Ulmer et al. cited supra).
The inactivation of DNAse I due to the binding to G-actin is a major problem for the therapeutic use of DNAse I. Attempts to circumvent this problem have been made, especially by trying to decrease the DNAse actin interaction by using alternative forms of DNAse. Examples of such attempts are the use of site directed mutagenesis of DNAse I in order to decrease its binding affinity for actin (International patent publication WO 96/26279), the use of DNAse I having a lower pH optimum but which does not bind to actin (International patent publication WO98/16659) or the use of novel DNAse molecules having a lower affinity for actin (International patent publication WO97/28266).
As an alternative, the formation of free G actin is prevented by some actin binding proteins. For instance, U.S. Pat. No. 5,656,589 describes the administration of plasma gelsolin and vitamin D binding protein to patients with pulmonary diseases. Gelsolin has three actin binding sites and can bind to monomeric and polymeric actin. It therefore has as such a viscosity decreasing effect by severing actin filaments and has a beneficial effect on the DNAse I treatment by binding the inhibitory G actin before the latter binds to DNAse I. U.S. Pat. No. 5,464,817 describes the detrimental influence of contaminants associated with actin binding proteins, such as non-actin binding peptides, carbohydrates, glycosylated peptides, lipids, membranes and others. These contaminants can be harmful as such when administered together with the pharmaceutical compound or can interfere with the desired therapeutic effects.
Therefore there is a constant need in the art for improving the performance of DNAse I in the treatment of pulmonary diseases such as CF. Since most of the above-described solutions to this problem involve the administration of proteins which, as is well known to the skilled person, encounter stability problems and can provoke immune reactions, there is also a need for a more simple and less expensive solution to the above problem.
Other diseases are also concerned by an excess of G-actin or a decrease in intracellular F-actin. An example of such diseases is the Wiskott Aldrich Syndrome (hereinafter WAS) where a mutation in the X chromosome in the gene for WAS protein (hereinafter WASP) results in an X-linked hereditary disease characterised by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections and bloody diarrhoea. Death usually occurs before the age of 10 years according to Derry et al. in Hum. Molec. Genet. (1995) 4:1127-1135 and Derry et al. in Cell (1994) 78:635-644. The WASP protein was shown to regulate the intracellular actin network. Defective WASP results in a lower level of F-actin (Fachetti et al. in J. Pathol. (1998) 185:99-107; Gallego et al. in Blood (1997) 90:3089-3097). Therefore there is a need in the art for promoting F actin formation by shifting the equilibrium between G actin and F actin for the prevention or treatment of such diseases induced by or inducing an excess of G actin.
Systemic Lupus Erythematosus (SLE) is characterized by the production of pathogenic autoantibodies against nucleoprotein antigens and double stranded DNA (dsDNA). SLE is a multifactorial and polygenic disease. It is a chronic, remitting, relapsing, inflammatory, and often febrile multisystemic disorder of connective tissue, acute or insidious at onset, is characterized principally by involvement of the skin, joints, kidneys, and serosal membranes. Of unknown aetiology, lupus erythematosus is thought to represent a failure of the regulatory mechanisms of the autoimmune system. (see Online Mendelian Inheritance in Man, OMIM (TM) Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.)).
In a lupus prone mouse model it has been shown that rhDNase I administration delays the development of dsDNA antibodies, reduces proteinuria and delays mortality. The pharmacokinetics and the activity of disease markers after administration of rhDNAse I were determined by Davis et al. in Lupus (1999) 8:68-76 and Prince et al. in Clin. Exp. Immunol. (1998) 113:289-296. These studies show that rhDNAse I is able to degrade DNA in DNA/antibody complexes and that inhibitors are present in the serum of patients.
The use of magnesium as a therapeutic agent has been reviewed by Swain et al. in South Med. J. (1999) 92:1040-1047. Magnesium has long been used as an ingredient in laxatives and antacids. Intravenous magnesium also is effective for the suppression of ventricular ectopy in the hospital setting and is a first-line agent for torsades de pointes. It is less clear whether it is useful in patients with congestive heart failure or acute myocardial infarction. Although effective for treatment of pre-eclampsia and eclampsia, its use in the termination of pre-term labor has recently been questioned. In asthma and chronic lung disease, intravenous magnesium may also be useful. Finally, magnesium may have a role in the prevention and treatment of vascular headaches.
In allergic diseases such as asthma, magnesium plays a role in the inhibition of histamine release from mast cells or in the relaxation of smooth lung muscle (Nannini et al. in Chest (1997) 111:858-861). In this respect, magnesium is not interfering with externally supplied therapeutic compounds.